The nuclear immunofluorescence signal intensity of phospho-H2AX was quantified by automated analysis using the Fiji software. To do so, images with the same exposure time were taken with the Axio Cam MRc/503 camera installed on the Axio Scope A1 microscope (Zeiss). As a nuclear counterstain, DAPI was used to define the regions of interest prior to the measurement of the mean intensity of the Alexa Fluor 568 staining (phospho-H2AX). At least 200 cells were subjected to analysis and quantification in each sample.
Axio cam mrc 503 camera
Manufactured by Zeiss
The Axio Cam MRc/503 camera is a high-performance microscope camera designed by Zeiss. It features a high-resolution sensor and advanced image processing capabilities. The camera is capable of capturing detailed images and supports a range of microscopy techniques.
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Lab products found in correlation
4 protocols using axio cam mrc 503 camera
1
Automated Quantification of Nuclear DNA Damage
2
Automated Quantification of DNA/RNA Structures
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Images were acquired (same exposure time for all images for each fluorescent channel per experiment) with Axio Scope A1 microscope (Zeiss) equipped with an Axio Cam MRc/503 camera.
The Fiji software was used for automated analysis and quantification of nuclear S9.6 or EdU staining. DAPI staining was used to identify regions of interest (nuclei) prior to measuring mean intensity of the Alexa Fluor 488 staining (S9.6), Alexa Flour 488 picolyl-azide or Alexa Fluor 594 picolyl-azide (EdU). At least 200 cells were subjected to analysis and quantification.
The Fiji software was used for automated analysis and quantification of nuclear S9.6 or EdU staining. DAPI staining was used to identify regions of interest (nuclei) prior to measuring mean intensity of the Alexa Fluor 488 staining (S9.6), Alexa Flour 488 picolyl-azide or Alexa Fluor 594 picolyl-azide (EdU). At least 200 cells were subjected to analysis and quantification.
3
Double-Blinded DNA Fiber Imaging
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To avoid bias, data acquisition and analysis were conducted in a double-blinded manner where identities of the samples were blinded prior to imaging and analysis.
Whenever possible, a minimum of 100 DNA fiber structures were visualized with fluorescence microscopy (Axio Scope A1 microscope (Zeiss) equipped with an Axio Cam MRc/503 camera) and analyzed.
For the 7-label fiber assay, the number of labels incorporated was counted using the cell counter plugin on Fiji. Fork stalling was then calculated by dividing the number of tracks with less than all seven labels by the total number of tracks and converted into percentage. The length of the second to third label was measured to determine the replication progression for the 7-label fiber assay. The Fiji software (RRID:SCR_002285) ( 22) was used to measure the labeled tracks in pixels and converted to micrometers using the conversion factor of 1 µm = 5.7 pixels (as determined by measuring scale bar under the same microscope settings) and then to kilo base (kb) using the conversion factor 1 µm = 2.59 kb. Rate of fork progression was calculated by dividing the number of bases by the labeling time of the track.
For the 2-label fiber assays, fibers were analyzed for their IdU track length and IdU fork progression rate calculated as described.
Whenever possible, a minimum of 100 DNA fiber structures were visualized with fluorescence microscopy (Axio Scope A1 microscope (Zeiss) equipped with an Axio Cam MRc/503 camera) and analyzed.
For the 7-label fiber assay, the number of labels incorporated was counted using the cell counter plugin on Fiji. Fork stalling was then calculated by dividing the number of tracks with less than all seven labels by the total number of tracks and converted into percentage. The length of the second to third label was measured to determine the replication progression for the 7-label fiber assay. The Fiji software (RRID:SCR_002285) ( 22) was used to measure the labeled tracks in pixels and converted to micrometers using the conversion factor of 1 µm = 5.7 pixels (as determined by measuring scale bar under the same microscope settings) and then to kilo base (kb) using the conversion factor 1 µm = 2.59 kb. Rate of fork progression was calculated by dividing the number of bases by the labeling time of the track.
For the 2-label fiber assays, fibers were analyzed for their IdU track length and IdU fork progression rate calculated as described.
4
Automated Nuclear S9 Quantification
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Images were acquired (same exposure time for all images for each fluorescent channel per experiment) with Axio Scope A1 microscope (Zeiss) equipped with an Axio Cam MRc/503 camera.
The Fiji software was used for automated analysis and quantification of nuclear S9.
The Fiji software was used for automated analysis and quantification of nuclear S9.
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